Cyanobacteria are among the most ancient organisms on Earth and have evolved chemical weapons for defensive purposes, which we are exploiting for anticancer drug discovery. Our past research has exemplified that marine cyanobacteria contain compounds with exceptionally potent activity and/or possess unusual or first-in-class inhibitors with novel mechanisms of action. However, cyanobacteria are also notorious for producing toxins detrimental to human health and other general cytotoxins. Our proposal addresses main issues in natural products drug discovery, including appropriate sample selection and prioritization for those compounds with promising therapeutic potential and cancer selectivity through innovative dereplication strategies and unique complementary in vitro cellular and in vivo organismal assay sets directed towards key cancer-related pathways. Targeted pathways are related to growth factor receptor activity and angiogenesis, resistance and metastasis, all of which are major areas of concern in cancer research. We have developed new chemical, biochemical and genetic tools to specifically probe novel mechanisms of action that we recently discovered and to identify modulators of these pathways. First, we will carry out field collections of marine cyanobacteria and will subject their extracts to taxonomy- and LC-MS/MS based dereplication strategies in combination with cytotoxicity assays. Prioritized cyanobacteria will be cultured and fraction libraries generated. Second, we will implement a rational screening strategy for selective cytotoxins by assessing differential cytotoxicity of fraction libraries using various suitable human cell-based models. Specifically, we will screen for agents with selective activity against colon cancer cells over the corresponding normal cells, in a mechanistically-unbiased approach. More targeted, we will screen for antiangiogenic agents that exert their activity through a mechanism we recently validated, involving simultaneous downregulation of receptors and growth factors. Through the use of our newly created unique isogenic screening system we then aim to discover novel dual inhibitors of the transcription factors HIF-1 and HIF-2, which have promise for combination therapy with anti-angiogenic agents. Prioritized fractions will be subjected to bioassay-guided isolation and structures will be determined. Third, we have discovered and characterized a novel mechanism to prevent metastasis and developed suitable zebrafish in vivo models to screen for anti-invasive agents that modulate tumor suppressor E-cadherin expression and localization to the cell membrane when applied as single agents or in combination with a synergizing cyanobacterial compound we already discovered. Anti-invasive properties of compounds will be assessed in a new zebrafish tumor model at the single-cell level by monitoring cell dissemination, invasion and metastasis. Fourth, we will mechanistically characterize the identified selective agents to pinpoint the molecular changes induced in the cancer cell and to determine potential direct targets.